scholarly journals Molecular cloning, expression and characterization of Bmserpin-2 gene from Bombyx mori.

2009 ◽  
Vol 56 (4) ◽  
Author(s):  
Ye Pan ◽  
Hengchuan Xia ◽  
Peng Lü ◽  
KePing Chen ◽  
Qin Yao ◽  
...  
Keyword(s):  
Bombyx Mori ◽  
Developmental Stages ◽  
Pcr Analysis ◽  
Post Inoculation ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Reading Frame ◽  
E Coli ◽  
Real Time Quantitative Pcr ◽  
Localization Analysis

Serpins are a broadly distributed family of protease inhibitors. In this study, the gene encoding Bombyx mori serpin-2 (Bmserpin-2) was cloned and expressed in E. coli. The Bmserpin-2 cDNA contains a 1125 bp open reading frame (ORF). The deduced protein has 374 amino-acid residues, contains a conserved SERPIN domain and shares extensive homology with other invertebrate serpins. RT-PCR analysis showed that Bmserpin-2 was expressed in all developmental stages of B. mori larvae and various larval tissues. Subcellular localization analysis indicated that Bmserpin-2 protein was located in the cytoplasm. Interestingly, real-time quantitative PCR revealed that the expression of Bmserpin-2 in the midgut of susceptible B. mori strain 306 significantly increased at 72 hours post inoculation (hpi) when infected with BmNPV. However, there was no significant increase of the Bmserpin-2 expression in resistant strain NB infected with BmNPV. Thus, our data indicates that Bmserpin-2 may be involved in B. mori antiviral response.

Molecular Cloning, Expression and Characterization of BmIDGF Gene from Bombyx mori

2010 ◽  
Vol 65 (3-4) ◽  
pp. 277-283 ◽  
Author(s):  
Ye Pan ◽  
Keping Chen ◽  
Hengchuan Xia ◽  
Qin Yao ◽  
Lu Gao ◽  
...  
Keyword(s):  
Bombyx Mori ◽  
Developmental Stages ◽  
Pcr Analysis ◽  
Amino Acid Residues ◽  
Glycosyl Hydrolases ◽  
Silkworm Larvae ◽  
Group V ◽  
E Coli ◽  
Sds Page ◽  
Localization Analysis

Imaginal disc growth factors (IDGF) play a key role in insect development, but their mechanism remains unclear. In this study, we cloned a novel IDGF gene in Bombyx mori and designated it as BmIDGF. We found that the BmIDGF gene contains eight exons and seven introns, encoding a peptide of 434 amino-acid residues. The protein was predicted to contain one conserved motif of the glycosyl hydrolases family 18 and fall into group V chitinases. Sequence alignment showed that BmIDGF shares extensive homology with other invertebrate IDGF. RT-PCR analysis showed that BmIDGF is expressed in all developmental stages of silkworm larvae and various larvae tissues, which was further confirmed by Western blot analysis. Subcellular localization analysis indicated that BmIDGF is located in the extracellular space. We also successfully expressed it in E. coli and further characterized it by SDS-PAGE and mass spectrometry. Taken together, our data suggests that BmIDGF is a chitinase-like extracellular protein, and provides an excellent platform for subsequent studies on its enzyme activity and role in B. mori development

scholarly journals Cloning of Acyl-ACP Thioesterase FatA fromArachis hypogaeaL. and Its Expression inEscherichia coli

2012 ◽  
Vol 2012 ◽  
pp. 1-5 ◽  
Author(s):  
Gao Chen ◽  
Zhen-ying Peng ◽  
Lei Shan ◽  
Ning Xuan ◽  
Gui-ying Tang ◽  
...  
Keyword(s):  
Oleic Acid ◽  
Palmitoleic Acid ◽  
Membrane Lipid ◽  
Pcr Analysis ◽  
Fatty Acid Profiles ◽  
Reading Frame ◽  
E Coli ◽  
Real Time Quantitative Pcr ◽  
Immature Seeds ◽  
Expression Inescherichia Coli

In this study, a full-length cDNA of the acyl-ACP thioesterase,AhFatA, was cloned from developing seeds ofArachis hypogaeaL. by 3′-RACE. Sequence analysis showed that the open reading frame encodes a peptide of 372 amino acids and has 50–70% identity with FatA from other plants. Real-time quantitative PCR analysis revealed thatAhFatA was expressed in all tissues ofA. hypogaeaL., but most strongly in the immature seeds harvested at 60 days after pegging. Heterologous expression ofAhFatA inEscherichia coliaffected bacterial growth and changed the fatty acid profiles of the membrane lipid, resulting in directed accumulation towards palmitoleic acid and oleic acid. These results indicate that AhFatA is at least partially responsible for determining the high palmitoleic acid and oleic acid composition ofE. coli.

scholarly journals Cloning and Sequencing of the Gene Encoding Toho-2, a Class A β-Lactamase Preferentially Inhibited by Tazobactam

1998 ◽  
Vol 42 (5) ◽  
pp. 1181-1186 ◽  
Author(s):  
Ling Ma ◽  
Yoshikazu Ishii ◽  
Masaji Ishiguro ◽  
Hiroshi Matsuzawa ◽  
Keizo Yamaguchi
Keyword(s):  
Amino Acid ◽  
Acid Resistance ◽  
Penicillin G ◽  
Amino Acid Residues ◽  
Incompatibility Group ◽  
Gene Encoding ◽  
Reading Frame ◽  
E Coli ◽  
Class A ◽  
Hydrolysis Rates

ABSTRACT Escherichia coli TUM1083, which is resistant to ampicillin, carbenicillin, cephaloridine, cephalothin, piperacillin, cefuzonam, and aztreonam while being sensitive to cefoxitin, moxalactam, cefmetazole, ceftazidime, and imipenem, was isolated from the urine of a patient treated with β-lactam antibiotics. The β-lactamase (Toho-2) purified from the bacteria hydrolyzed β-lactam antibiotics such as penicillin G, carbenicillin, cephaloridine, cefoxitin, cefotaxime, ceftazidime, and aztreonam and especially had increased relative hydrolysis rates for cephalothin, cephaloridine, cefotaxime, and ceftizoxime. Different from other extended-spectrum β-lactamases, Toho-2 was inhibited 16-fold better by the β-lactamase inhibitor tazobactam than by clavulanic acid. Resistance to β-lactams was transferred by conjugation from E. coliTUM1083 to E. coli ML4909, and the transferred plasmid was about 54.4 kbp, belonging to the incompatibility group IncFII. The cefotaxime resistance gene for Toho-2 was subcloned from the 54.4-kbp plasmid. The sequence of the gene was determined, and the open reading frame of the gene was found to consist of 981 bases. The nucleotide sequence of the gene (DDBJ accession no. D89862) designated asbla toho was found to have 76.3% identity to class A β-lactamase CTX-M-2 and 76.2% identity to Toho-1. It has 55.9% identity to SHV-1 β-lactamase and 47.5% identity to TEM-1 β-lactamase. Therefore, the newly isolated β-lactamase designated as Toho-2 produced by E. coli TUM1083 is categorized as an enzyme similar to Toho-1 group β-lactamases rather than to mutants of TEM or SHV enzymes. According to the amino acid sequence deduced from the DNA sequence, the precursor consisted of 327 amino acid residues. Comparison of Toho-2 with other β-lactamase (non-Toho-1 group) suggests that the substitutions of threonine for Arg-244 and arginine for Asn-276 are important for the extension of the substrate specificity.

scholarly journals Characterization of a C-Type Lectin Domain-Containing Protein with Antibacterial Activity from Pacific Abalone (Haliotis discus hannai)

2022 ◽  
Vol 23 (2) ◽  
pp. 698
Author(s):  
Mi-Jin Choi ◽  
Yeo Reum Kim ◽  
Nam Gyu Park ◽  
Cheorl-Ho Kim ◽  
Young Dae Oh ◽  
...  
Keyword(s):  
Defense Mechanism ◽  
Developmental Stages ◽  
Haliotis Discus Hannai ◽  
Gene Encoding ◽  
Pacific Abalone ◽  
Reading Frame ◽  
Lectin Domain ◽  
Haliotis Discus ◽  
Abalone Haliotis Discus ◽  
Bacterial Agglutination

Genes that influence the growth of Pacific abalone (Haliotis discus hannai) may improve the productivity of the aquaculture industry. Previous research demonstrated that the differential expression of a gene encoding a C-type lectin domain-containing protein (CTLD) was associated with a faster growth in Pacific abalone. We analyzed this gene and identified an open reading frame that consisted of 145 amino acids. The sequence showed a significant homology to other genes that encode CTLDs in the genus Haliotis. Expression profiling analysis at different developmental stages and from various tissues showed that the gene was first expressed at approximately 50 days after fertilization (shell length of 2.47 ± 0.13 mm). In adult Pacific abalone, the gene was strongly expressed in the epipodium, gill, and mantle. Recombinant Pacific abalone CTLD purified from Escherichia coli exhibited antimicrobial activity against several Gram-positive bacteria (Bacillus subtilis, Streptococcus iniae, and Lactococcus garvieae) and Gram-negative bacteria (Vibrio alginolyticus and Vibrio harveyi). We also performed bacterial agglutination assays in the presence of Ca2+, as well as bacterial binding assays in the presence of the detergent dodecyl maltoside. Incubation with E. coli and B. subtilis cells suggested that the CTLD stimulated Ca2+-dependent bacterial agglutination. Our results suggest that this novel Pacific abalone CTLD is important for the pathogen recognition in the gastropod host defense mechanism.

scholarly journals Dissemination of CTX-M-Type β-Lactamases among Clinical Isolates of Enterobacteriaceae in Paris, France

2004 ◽  
Vol 48 (4) ◽  
pp. 1249-1255 ◽  
Author(s):  
C. Eckert ◽  
V. Gautier ◽  
M. Saladin-Allard ◽  
N. Hidri ◽  
C. Verdet ◽  
...  
Keyword(s):  
Insertion Sequence ◽  
Dna Fingerprint ◽  
Clinical Isolates ◽  
Pcr Analysis ◽  
Reading Frame ◽  
E Coli ◽  
Paris Area ◽  
The Family ◽  
Genes Encoding ◽  
Cephalosporin Resistance

ABSTRACT We analyzed 19 clinical isolates of the family Enterobacteriaceae (16 Escherichia coli isolates and 3 Klebsiella pneumoniae isolates) collected from four different hospitals in Paris, France, from 2000 to 2002. These strains had a particular extended-spectrum cephalosporin resistance profile characterized by a higher level of resistance to cefotaxime and aztreonam than to ceftazidime. The bla CTX-M genes encoding these β-lactamases were involved in this resistance, with a predominance of bla CTX-M-15. Ten of the 19 isolates produced both TEM-1- and CTX-M-type enzymes. One strain (E. coli TN13) expressed CMY-2, TEM-1, and CTX-M-14. bla CTX-M genes were found on large plasmids. In 15 cases the same insertion sequence, ISEcp1, was located upstream of the 5′ end of the bla CTX-M gene. In one case we identified an insertion sequence designated IS26. Examination of the other three bla CTX-M genes by cloning, sequencing, and PCR analysis revealed the presence of a complex sul1-type integron that includes open reading frame ORF513, which carries the bla gene and the surrounding DNA. Five isolates had the same plasmid DNA fingerprint, suggesting clonal dissemination of CTX-M-15-producing strains in the Paris area.

Cloning and Expression of a Novel Protease with Fibrinolytic Activity from Arenicola cristata

2014 ◽  
Vol 998-999 ◽  
pp. 210-213
Author(s):  
Chun Ling Zhao ◽  
Wen Jing Yu ◽  
Ji Yu Ju
Keyword(s):  
Amino Acid ◽  
Recombinant Protein ◽  
Active Form ◽  
Cloning And Expression ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Reading Frame ◽  
Arenicola Cristata ◽  
Fibrin Plate ◽  
Serine Protease Family

cDNA of a novel protease, designated as AFEI, was cloned from digestive tract of Arenicola cristata by RACE. The cDNA of AFEIcomprised 897bp and an open reading frame that encoded polypeptides of 264 amino acid residues. AFEIshowed similarity to serine protease family and contained the conserved catalytic amino acid residues. The gene encoding the active form of AFEIwas expressed in E.coli and the purified recombinant protein could dissolve an artificial fibrin plate with plasminogen, which indicated the recombinant protein might be a plasminogen activator for thrombosis therapy.

Molecular Cloning and Characterization of a cDNA Encoding a Transferrin Homolog from Bombyx mori

1999 ◽  
Vol 380 (12) ◽  
pp. 1455-1459 ◽  
Author(s):  
Eun Young Yun ◽  
Seok Woo Kang ◽  
Jae Sam Hwang ◽  
Tae Won Goo ◽  
Sang Hyun Kim ◽  
...  
Keyword(s):  
Bombyx Mori ◽  
Cdna Sequence ◽  
The Self ◽  
Open Reading Frame ◽  
Iron Binding ◽  
Defense System ◽  
Reading Frame ◽  
E Coli ◽  
Self Defense

Abstract We isolated a cDNA representing a message that was strongly induced by injection with E. coli in Bombyx mori. The 2160 bp cDNA has an open reading frame of 644 amino acids and the deduced product a predicted molecular mass of 71 kDa. The cDNA sequence shared high homology with the transferrins known so far, and its deduced peptide had unique features of transferrins, that is, sites of cystein residues and iron binding. We suggest that the B. mori transferrin plays an important role in the self-defense system.

Molecular Cloning and Characterization of cDNA Encoding Fibrinolytic Enzyme-3 from Earthworm Eisenia foetida

2004 ◽  
Vol 36 (4) ◽  
pp. 303-308 ◽  
Author(s):  
Guo-Qing Dong ◽  
Xiao-Ling Yuan ◽  
Ya-Jun Shan ◽  
Zhen-Hu Zhao ◽  
Jia-Pei Chen ◽  
...  
Keyword(s):  
Inclusion Bodies ◽  
Fibrinolytic Enzyme ◽  
Eisenia Foetida ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Native Form ◽  
Reading Frame ◽  
Fibrin Plate ◽  
Earthworm Fibrinolytic Enzyme ◽  
High Degree

Abstract The earthworm fibrinolytic enzyme-3 (EFE-3, GenBank accession No: AY438622), from the earthworm Eisenia foetida, is a component of earthworm fibrinolytic enzymes. In this study, cDNA encoding the EFE-3 was cloned by RT-PCR. The cDNA contained an open reading frame of 741 nucleotides, which encoded a deduced protein of 247 amino acid residues, including signal sequences. EFE-3 showed a high degree of homology to earthworm (Lumbricus rebullus) proteases F-III-1, F-III-2, and bovine trypsin. The recombinant EFE-3 was expressed in E. coli as inclusion bodies, and the gene encoding the native form of EFE-3 was expressed in COS-7 cells in the medium. Both the refolding product of inclusion bodies and the secreted protease could dissolve the artificial fibrin plate.

scholarly journals Screening Potential Reference Genes in Tuta absoluta with Real-Time Quantitative PCR Analysis under Different Experimental Conditions

Genes ◽  
2021 ◽  
Vol 12 (8) ◽  
pp. 1253
Author(s):  
An-Pei Yang ◽  
Yu-Sheng Wang ◽  
Cong Huang ◽  
Zhi-Chuang Lv ◽  
Wan-Xue Liu ◽  
...  
Keyword(s):  
Reference Genes ◽  
Molecular Mechanisms ◽  
Developmental Stages ◽  
Tuta Absoluta ◽  
Pcr Analysis ◽  
Internal Reference ◽  
Experimental Conditions ◽  
Real Time Quantitative Pcr ◽  
Adult Tissues ◽  
Induced Stresses

Tuta absoluta is one of the most significant invasive pests affecting tomato plants worldwide. RT-qPCR has emerged as one of the most sensitive and accurate methods for detecting gene expression data. The screening of stable internal reference genes is the most critical step for studying the molecular mechanisms of environmental adaptability. The stable reference genes expressed in T. absoluta under specific experimental conditions have not yet been clarified. In this study, seven candidate reference genes (RPL27, RPS13, RPS15, EF1-α, TUB, TBP, and β-actin) and their optimal numbers were evaluated under biotic (developmental stages and adult tissues) and abiotic (insecticide, temperature, and plant VOC) conditions using four software programs. Our results identified the following reference genes and numbers as optimal: three genes (EF1-α, RPS13, and RPL27) for different developmental stages (egg, larva, pupa, unmated adult), two genes (RPS13 and TBP) for adult tissues (antenna, head, thorax, abdomen, leg), two genes (TBP and RPS13) for insecticides (Bacillus thuringiensis, chlorpyrifos, abamectin-aminomethyl, and chlorantraniliprole), two genes (RPL27 and TUB) for temperature-induced stresses (0, 25, and 40 °C), and two genes (RPS13 and TUB) for VOC-induced stresses (nonanal, α-phellandrene, and tomato leaves). Our results provide a reference for selecting appropriate reference genes for further study of the functional genes of T. absoluta under different experimental conditions.

scholarly journals Isolation, Characterization, and Expression Analysis of the MaMDH Gene in Banana

HortScience ◽  
2013 ◽  
Vol 48 (5) ◽  
pp. 614-619
Author(s):  
Cai-Hong Jia ◽  
Ju-Hua Liu ◽  
Zhi-Qiang Jin ◽  
Qiu-Ju Deng ◽  
Jian-Bin Zhang ◽  
...  
Keyword(s):  
Real Time ◽  
Quantitative Polymerase Chain Reaction ◽  
Expression Patterns ◽  
Ethylene Biosynthesis ◽  
Pcr Analysis ◽  
Malic Dehydrogenase ◽  
Reading Frame ◽  
Expression Levels ◽  
Post Harvest ◽  
Real Time Quantitative Pcr

A full-length cDNA isolated from banana (Musa acuminata L. AAA group) fruit was named MaMDH, containing an open reading frame encoding 332 amino acids that represents the gene for cytoplasmic malic dehydrogenase (MDH). Sequence analysis showed that MaMDH shares high similarity with MDHs from castor bean (XP_002533463), tobacco (CAC12826), peach (AAL11502), and chickpeas (CAC10208). Real-time quantitative polymerase chain reaction (PCR) analysis of MaMDH spatial expression showed that it was expressed in all organs examined: roots, rhizomes, leaves, flowers, and fruits. The expression was the highest in flowers followed by the fruits and roots, whereas the rhizomes and leaves displayed the lowest expression levels. Real-time quantitative PCR revealed that MaMDH exhibited differential expression patterns in post-harvest banana fruits correlating with ethylene biosynthesis. In naturally ripened banana fruits, MaMDH expression was in accordance with ethylene biosynthesis. In accordance, for banana fruits treated with the ethylene analog 1-methylclopropene (1-MCP), MaMDH expression levels were inhibited and remained constant. After treatment with ethylene, MaMDH expression in banana fruits significantly increased with ethylene biosynthesis and peaked 3 days after harvest, which was 11 days earlier than that in naturally ripened banana fruits. These results suggest that MaMDH expression is induced by ethylene to regulate post-harvest banana fruits ripening.

Sign in / Sign up

Export Citation Format

Share Document